There is a recognized need for antiviral agents to combat the threat to millions of humans from refractory viruses such as the retrovirus believed to cause Acquired Immunodeficiency Syndrome ("AIDS") disease.
Currently AIDS disease is killing thousands of humans throughout the world, and wreaking psychological havoc in its wake. AIDS is caused by a retrovirus, human T4 lymphotropic virus (HTLV-III), more recently designated as human immunodeficiency virus or "HIV". This virus has the capacity to replicate within the cells of the human immune system to the destruction of helper/inducer (T4+ or CD4+) T cells. Retroviruses are a type of virus that replicates through a DNA intermediate, i.e. during replication genetic information is transferred from RNA to DNA, in a reverse or "retro" direction. This step is catalyzed by reverse transcriptase, a viral DNA polymerase. Many retroviruses are capable of inducing neoplastic transformation in infected target cells, although HIV is an exception to this rule. AIDS disease manifests as a loss of delayed-type cutaneous hypersensitivity reactions, a loss of certain in vitro T-cell proliferative responses, excessive immunoglobulin production by B cells, and a loss of in vitro cytotoxic T-cell responses.
Although the immunodeficiency state of AIDS disease is generally progressive and fatal, the effect of infection with HIV varies. While AIDS disease results in a severe suppression of the immune system manifesting a number of "opportunistic" infections (e.g. caused by microorganisims that rarely cause disease in individuals with normal immune systems) and cancers, some individuals infected with HIV may not develop AIDS disease. AIDS-related complex (ARC) is a condition characterized by fevers, diarrhea and swollen lymph nodes in approximately 50% of individuals infected with HIV. In both AIDS and ARC, the HIV may be present in cells for prolonged periods of time (months and even years) without causing cell death.
Although considerable effort and expense is being devoted to developing an effective treatment for AIDS, the disease has failed to respond significantly to existing therapies. Therapeutic intervention for the treatment of pathogenic human retroviruses such as AIDS has typically targeted different stages in the life cycle of the HIV virus for administration of antiviral agents. A certain amount of success has been met by attempts to prevent viral replication, for example using reverse transcriptase inhibitors such as the 2'3'-dideoxy-nucleoside analog 3'-azido 3'-deoxythymidine (AZT). Other approaches include the use of antibodies to the virus or cell receptor, drugs that block virus fusion with a target cell or interfere with viral uncoating; inhibitors of RNase H activity and interferons. The testing of anti-retroviral agents has used in vitro screening systems to determine whether the agent can inhibit the replication and T-cell killing activity of the virus (Mitsuya et al, Nature, 325:773-778 (1987)).
L-canavanine is an amino acid found in the Lotoidae, a major subfamily of the Leguminosae, and it is the principal free amino acid of numerous legumes (Rosenthal, Qt. Rev. Biol., 52:155-178 (1977)). It is a guanidinooxy analog of the amino acid L-Arginine, having the formula H.sub.2 N--C(.dbd.NH)--NH--O--CH.sub.2 --CH.sub.2 --CH(NH.sub.2)COOH. The guanidinooxy group of canavanine has a pK of 8.2 as compared to a pK of 10.8 for the guanidino group of arginine. This decreased basicity may effect the activity and structural properties of proteins containing canavanine as demonstrated by the appearance of T4 phage possessing giant polyheads as a result of the incorporation of canavanine (Chapman et al., Virology 54:245-261 (1973)).
The antimetabolic properties of canavanine have been observed in vitro in many types of microorganisms, plants, insects and animal cells and appear to be due to a marked inhibition of RNA and DNA synthesis, with little effect on net protein synthesis. The inhibitory effects are readily reversible or prevented by the administration of arginine (Id.). Although the mechanism is uncertain, the basis of toxicity is believed to stem from the incorporation of canavanine into proteins, as has been demonstrated in several organisms including Escherichia coli (Schachtele et al., J. Mol. Biol., 14:474-489 (1965); adenovirus (Neurath et al., Biochem. Biophys. Res. Commun., 41:1509-1517 (1970); Chlamydomonas reinhardi (McMahon et al., J. Gen. Microbiol. 73:239-250 (1972)); Walker carcinosarcoma 256 cells (Kruse et al., Cancer Res, 19:122-125 (1959)) and tobacco hornworm larvae (Cahlman et al, J. Insect Physiol., 22:265-271 (1976)). L-canavanine is also known to inhibit cellular and viral replication rapidly (Bell, J. Gen. Virol., 22:319-330 (1974)) and to promote more rapid degradation of proteins which have been structurally modified as a result of its incorporation (Prouty, J. Cell Physiol., 88:371-382 (1976) and Goldberg et al., part 2, Ann. Rev. Biochem. 45:747-803 (1976)).
Green et al. (Cancer Research 40:535-537 (1980)) demonstrated that L-canavanine possesses antitumor activity in leukemic mice. L-canavanine was found to selectively inhibit DNA synthesis by L1210 leukemic cells as determined from ascites fluid, and to significantly prolong the lifespan of mice bearing these cells. These results indicate that L-canavanine selectively kills L1210 leukemic cells in mice.
Subsequently, L-canavanine was shown to exhibit selective cytotoxicity on chemically transformed derivatives of normal Madin-Darby canine kidney (MDCK) epithelial cells (Berjis et al., Interactions, 60:305-315 (1986)). Under conditions where the canavanine reversibly arrested growth of the normal MDCK cells, more than 90% of the tumorigenic MDCK-T cells were killed. The selective toxicity was not due to any difference in growth rates of the two cell types or from inhibition of protein synthesis or DNA replication.
Recently, the inhibitory effects of L-canavanine on the growth of a solid animal tumor in vivo have been reported (Thomas et al., Cancer Research 46:2898-2903 (1986)). Reduction in tumor volume of rat colon tumor was observed in animals receiving 2 g/kg of L-canavanine for five days. Cumulative toxicity caused death in some of the animals at higher doses (3 g/kg for 9 days).
The toxicity of L-canavanine in mammals was explored by prolonged oral feeding of monkeys with the analog which resulted in the induction of biochemical effects characteristic of the autoimmune disease, systemic lupus erythematosus (Malinow et al., Science, 216:415-417 (1982)).
The present invention provides a method of using the amino acid analog L-canavanine to selectively kill mammalian cells infected with and producing virus and to treat disease caused by the virus.